1. Field of the Invention
This invention relates to methods and apparatus for selectively targeting specific cells within a mixed population of living cells. In particular, this invention relates to methods and apparatus for selectively identifying, and individually targeting with an energy beam, specific cells within a mixed cell population to induce a response in the targeted cells.
2. Description of the Related Art
The use of cellular therapies is growing rapidly, and is therefore becoming an important therapeutic modality in the practice of medicine. Unlike other therapies, cellular therapies achieve a long-lasting, and often permanent benefit through the use of living cells. Hematopoietic stem cell (HSC) (e.g., bone marrow or mobilized peripheral blood) transplantation is one example of a practiced, insurance-reimbursed cellular therapy. Many other cellular therapies are being developed, including immunotherapy for cancer and infectious diseases, chondrocyte therapy for cartilage defects, neuronal cell therapy for neurodegenerative diseases, and stem cell therapy for numerous indications. Many of these therapies require the removal of unwanted, detrimental cells for full efficacy to be realized.
Gene therapy is another active area of developing medicine that can influence the success of cellular therapy. Given the rapid advances in the understanding of the human genome, it is likely that many genes will be available for insertion into cells prior to transplantation into patients. However, obtaining efficient targeted delivery of genes into specific cells of interest has remained a difficult obstacle in the development of these therapies.
In the treatment of cancer, it has been found that high-dose chemotherapy and/or radiation therapy can be used to selectively kill rapidly dividing cancer cells in the body. Unfortunately, several other cell types in the body are also rapidly dividing, and in fact, the dose-limiting toxicity for most anti-cancer therapies is the killing of HSCs and progenitor cells in the bone marrow. HSC transplantation was developed as a therapy to rescue the hematopoietic system following anti-cancer treatments. Upon infusion, the HSCs and progenitor cells within the transplant selectively home to the bone marrow and engraft. This process is monitored clinically through daily blood cell counts. Once blood counts return to acceptable levels, usually within 20 to 30 days, the patient is considered engrafted and is released from the hospital.
HSC transplants have been traditionally performed with bone marrow, but mobilized peripheral blood (obtained via leukapheresis after growth factor or low-dose chemotherapy administration) has recently become the preferred source because it eliminates the need to harvest approximately one liter of bone marrow from the patient. In addition, HSCs from mobilized peripheral blood result in more rapid engraftment (8 to 15 days), leading to less critical patient care and earlier discharge from the hospital. HSC transplantation has become an established therapy for treating many diseases, such that over 45,000 procedures were performed worldwide in 1997.
HSC transplantation may be performed using either donor cells (allogeneic), or patient cells that have been harvested and cryopreserved prior to administration of high-dose anti-cancer therapy (autologous). Autologous transplants are widely used for treating a variety of diseases including breast cancer, Hodgkin""s and non-Hodgkin""s lymphomas, neuroblastoma, and multiple myeloma. The number of autologous transplants currently outnumbers allogeneic transplants by approximately a 2:1 ratio. This ratio is increasing further, mainly due to graft-versus-host disease (GVHD) complications associated with allogeneic transplants. One of the most significant problems with autologous transplants is the reintroduction of tumor cells to the patient along with the HSCs, because these tumor cells contribute to relapse of the original disease.
As a tumor grows, tumor cells eventually leave the original tumor site and migrate through the bloodstream to other locations in the body. This process, called tumor metastasis, results in the formation and growth of satellite tumors that greatly increase the severity of the disease. The presence of these metastatic tumor cells in the blood and other tissues, often including bone marrow, can create a significant problem for autologous transplantation. In fact, there is a very high probability that metastatic tumor cells will contaminate the harvested HSCs that are to be returned to the patient following anti-cancer therapy.
The presence of contaminating tumor cells in autologous bone marrow and mobilized peripheral blood harvests has been confirmed in numerous scientific studies. Tumor cell contamination has been repeatedly observed in patients with T-cell lymphoma, non-Hodgkin""s lymphoma, leukemia, neuroblastoma, lung cancer, breast cancer, etc. (Brugger, W., Bross, K. J., Glatt, M., Weber, F., Mertelsmann, R., and Kanz, L.: Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 83: 636-640, 1994; Gulati, S. C. and Acaba, L.: Rationale for purging in autologous stem cell transplantation. J.Hematotherapy 2: 467-471, 1993; Kvalheim, G., Holte, H., Jakobsen, E., and Kvaloy, S.: Immunomagnetic purging of lymphoma cells from autografts. J.Hematotherapy 5: 561-562, 1996; Mapara, M. Y., Kxc3x6rner, I. J., Hildebrandt, M., Bargou, R., Krahl, D., Reichardt, P., and Dxc3x6rken, B.: Monitoring of tumor cell purging after highly efficient immunomagnetic selection of CD34 cells from leukapheresis products in breast cancer patients: Comparison of immunocytochemical tumor cell staining and reverse transcriptase-polymerase chain reaction. Blood 89: 337-344, 1997; Paulus, U., Dreger, P., Viehmann, K., von Neuhoff, N., and Schmitz, N.: Purging peripheral blood progenitor cell grafts from lymphoma cells: Quantitative comparison of immunomagnetic CD34+ selection systems. Stem Cells 15: 297-304, 1997; Shpall, E. J. and Jones, R. B.: Release of tumor cells from bone marrow. Blood 83: 623-625, 1994; Vervoordeldonk, S. F., Merle, P. A., Behrendt, H., Steenbergen, E. J., van den Berg, H., van Wering, E. R., von dem Borne, A. E. G., van der Schoot, C. E., van Leeuwen, E. F., and Slaper-Cortenbach, I. C. M.: PCR-positivity in harvested bone marrow predicts relapse after transplantation with autologous purged bone marrow in children in second remission of precursor B-cell acute leukemia. Br.J.Haematol. 96: 395-402, 1997). In every study, all or nearly all of the patient samples analyzed were positive for tumor contamination. The level of tumor cell burden in these HSC harvests varied widely depending upon the type and stage of disease. Typical numbers indicate that tumor cells are present in the range of 3 to 3,000 tumor cells per million hematopoietic cells. Since the transplanted cell number is on the order of 10 billion hematopoietic cells, the total number of tumor cells in a transplant varies in the range of 30 thousand to 30 million. The reinfusion of this number of tumor cells in the HSC transplant following the patient""s anti-cancer therapy is of considerable clinical concern. In fact, animal models have shown that as few as 25 leukemia cells can establish a lethal tumor in 50% of mice, and these numbers extrapolate to 3500 cells in humans (Gulati, Acaba 1993).
Recent landmark studies have unambiguously shown that reinfused tumor cells do indeed contribute to disease relapse in humans (Rill, D. R., Santana, V. M., Roberts, W. M., Nilson, T., Bowman, L. C., Krance, R. A., Heslop, H. E., Moen, R. C., Ihle, J. N., and Brenner, M. K.: Direct demonstration that autologous bone marrow transplantation for solid tumors can return a multiplicity of tumorigenic cells. Blood 84: 380-383, 1994). This was proven by genetically marking the harvested cells prior to transplant, and then showing that the marker was detected in resurgent tumor cells in those patients who relapsed with disease. These data have been confirmed by other investigators (Deisseroth, A. B., Zu, Z., Claxton, D., Hanania, E. G., Fu, S., Ellerson, D., Goldberg, L., Thomas, M., Janicek, K., Anderson, W. F., Hester, J., Korbling, M., Durett, A., Moen, R., Berenson, R., Heimfeld, S., Hamer, J., Calver, L., Tibbits, P., Talpaz, M., Kantarjiam, H., Champlin, R., and Reading, C.: Genetic marking shows that Ph+ cells present in autologous transplants of chronic myelogenous leukemia (CML) contribute to relapse after autologous bone marrow transplantation in CML. Blood 83: 3068-3076, 1994), indicating that contaminating tumor cells in HSC transplants represent a real threat to patients undergoing autologous transplantation.
Subsequent detailed studies have now shown that the actual number of tumor cells reinfused in the transplant was correlated with the risk of relapse for acute lymphoblastic leukemia (Vervoordeldonk et al. 1997), non-Hodgkin""s lymphoma (Sharp, J. G., Joshi, S. S., Armitage, J. O., Bierman, P., Coccia, P. F., Harrington, D. S., Kessinger, A., Crouse, D. A., Mann, S. L., and Weisenburger, D. D.: Significance of detection of occult Non-Hodgkin""s Lymphoma in histologically uninvolved bone marrow by a culture technique. Blood 79: 1074-1080, 1992; Sharp, J. G., Kessinger, A., Mann, S., Crouse, D. A., Armitage, J. O., Bierman, P., and Weisenburger, D. D.: Outcome of high-dose therapy and autologous transplantation in non-Hodgkin""s lymphoma based on the presence of tumor in the marrow or infused hematopoietic harvest. J.Clin.Oncol. 14: 214-219, 1996), mantle cell lymphoma (Andersen, N. S., Donovan, J. W., Borus, J. S., Poor, C. M., Neuberg, D., Aster, J. C., Nadler, L. M., Freedman, A. S., and Gribben, J. G.: Failure of immunologic purging in mantle cell lymphoma assessed by polymerase chain reaction detection in minimal residual disease. Blood 90: 4212-4221, 1997), and breast cancer (Brockstein, B. E., Ross, A. A., Moss, T. J., Kahn, D. G., Hollingsworth, K., and Williams, S. F.: Tumor cell contamination of bone marrow harvest products: Clinical consequences in a cohort of advanced-stage breast cancer patients undergoing high-dose chemotherapy. J.Hematotherapy 5: 617-624, 1996; Fields, K. K., Elfenbein, G. J., Trudeau, W. L., Perkins, J. B., Janssen, W. E., and Moscinski, L. C.: Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation. J.Clin.Oncol. 14: 1868-1876, 1996; Schulze, R., Schulze, M., Wischnik, A., Ehnle, S., Doukas, K., Behr, W., Ehret, W., and Schlimok, G.: Tumor cell contamination of peripheral blood stem cell transplants and bone marrow in high-risk breast cancer patients. Bone Marrow Transplant. 19: 1223-1228, 1997; Vannucchi, A. M., Bosi, A., Glinz, S., Pacini, P., Linari, S., Saccardi, R., Alterini, R., Rigacci, L., Guidi, S., Lombarkini, L., Longo, G., Mariani, M. P., and Rossi-Ferrini, P.: Evaluation of breast tumour cell contamination in the bone marrow and leukapheresis collections by RT-PCR for cytokeratin-19 mRNA. Br.J.Haematol. 103: 610-617, 1998; Vredenburgh, J. J., Silva, O., Broadwater, G., Berry, D., DeSombre, K., Tyer, C., Petros, W. P., Peters, W. P., and Bast, J., R. C.: The significance of tumor contamination in the bone marrow from high-risk primary breast cancer patients treated with high-dose chemotherapy and hematopoietic support. Biol. Blood Marrow Transplant. 3: 91-97, 1997). One of these studies went even further, showing that the number of tumor cells infused was inversely correlated with the elapsed time to relapse (Vredenburgh et al. 1997). These data suggest that reducing the number of tumor cells in the transplant will lead to better outcomes for the patient.
In fact, one clinical study of NHL purging in 114 patients showed that disease-free survival (after a median 2-year follow-up) was substantially higher (93%) in the subset of patients that had all detectable tumor cells purged prior to transplant, as compared with those in which purging was unsuccessful (54%) (Gribben, J. G., Freedman, A. S., Neuberg, D., Roy, D. C., Blake, K. W., Woo, S. D., Grossbard, M. L., Rabinowe, S. N., Coral, F., Freeman, G. J., Ritz, J., and Nadler, L. M.: Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma. N.E.J.Med. 325: 1525-1533, 1991). In a recent update of this study, eight-year freedom-from-relapse was shown to be 83% in the subset of patients that had all detectable tumor cells purged, as compared to 19% in patients where purging was unsuccessful (Freedman, A. S., Neuberg, D., Mauch, P., Soiffer, R. J., Anderson, K. C., Fisher, D. C., Schlossman, R., Alyea, E. P., Takvorian, T., Jallow, H., Kuhlman, C., Ritz, J., Nadler, L. M., and Gribben, J. G.: Long-term follow-up of autologous bone marrow transplantation in patients with relapsed follicular lymphoma. Blood 94: 3325-3333, 1999). Therefore, the actual number of tumor cells in an HSC transplant, and the ability to reliably purge them, are of significant and growing importance in the delivery of HSC transplantation therapies for cancer patients.
Due to the known risk of tumor cell contamination in autologous transplantation, a number of methods have been proposed for removing contaminating tumor cells from harvested HSC populations. The basic principle underlying all purging methods is to remove or kill tumor cells while preserving the HSCs that are needed for hematopoietic reconstitution in the patient.
One method based on relatively non-specific adhesion differences of hematopoietic cells in deep bed filtration has been described by Dooley, et al. (Dooley, D. C., Xiao, M., Wickramasinghe, S. R., Oppenlander, B. K., and Castino, F.: A novel inexpensive technique for the removal of breast cancer cells from mobilized peripheral blood stem cell products. Blood 88: 252a, 1996). An elutriation method based on relatively non-specific cell size and density differences has been described by Wagner, et al. (Wagner, J. E., Collins, D., Fuller, S., Schain, L. R., Berson, A. E., Almici, C., Hall, M. A., Chen, K. E., Okarma, T. B., and Lebkowski, J. S.: Isolation of small, primitive human hematopoitic stem cells: Distribution of cell surface cytokine receptors and growth in SCID-Hu mice. Blood 86: 512-523, 1995). Preferential killing of tumor cells by hyperthermia has been described by Higuchi, et al. (Higuchi, W., Moriyama, Y., Kishi, K., Koike, T., Shibata, A., Shinada, S., Tada, I., and Miura, A.: Hematopoietic recovery in a patient with acute lymphoblastic leukemia after an autologous marrow graft purged by combined hyperthermia and interferon in vitro. Bone Marrow Transplant. 7: 163-166, 1991). These relatively non-specific methods reduce the number of tumor cells present, but a significant number are known to remain.
In another method, cytotoxic agents, such as 4-hydroxy-peroxy-cyclophosphamide (4-HC), were used to preferentially kill tumor cells in populations containing HSCs (Bird, J. M., Luger, S., Mangan, P., Edelstein, M., Silberstein, L., Powlis, W., Ball, J., Schultz, D. J., and Stadtmauer, E. A.: 4-Hydroperoxycyclophosphamide purged autologous bone marrow transplantation in non-Hodgkin""s lymphoma patients at high risk of none marrow involvement. BMT 18: 309-313, 1996). Unfortunately, collateral damage to normal HSCs was so severe that patient engraftment was delayed by as much as 59 days.
Another method employed preferential killing of tumor cells by exposing all cells to photoradiation in the presence of a light-sensitizing agent (e.g. merocyanide) (Gulliya, K. S. and Pervaiz, S.: Elimination of clonogenic tumor cells from HL-60, Daudi, and U-937 cell lines by laser photoradiation therapy: Implications for autologous bone marrow purging. Blood 73: 1059-1065, 1989). Although more tumor cells were killed than hematopoietic cells, some tumor cells still remained, and HSC damage was significant.
Another method for removing tumor cells from populations of hematopoietic cells involved immunoconjugating a toxic agent to an antibody having specificity for the tumor cells. In this system, antibodies were bound to chemotoxic agents, toxins, or radionucleides and then contacted with the total cell population. Unfortunately, not all of the tumor cells were killed by this treatment (Gribben et al. 1991; Robertson, M. J., Soiffer, R. J., Freedman, A. S., Rabinowe, S. L., Anderson, K. C., Ervin, T. J., Murray, C., Dear, K., Griffin, J. D., Nadler, L. M., and Ritz, J.: Human bone marrow depleted of CD33-positive cells mediates delayed but durable reconstitution of hematopoiesis: Clinical trial of MY9 monoclonal antibody-purged autografts for the treatment of acute myeloid leukemia. Blood 79: 2229-2236, 1992).
Some companies and physicians have attempted to purge malignant cells from populations of non-tumor cells using an immunoaffinity bead-based selection. In this procedure, the total cell population is contacted by immunoaffinity beads. For example, a first (positive) CD34-selection procedure enriches HSCs from the tumor cell-containing hematopoietic cell mixture. In some instances, a second (negative) immunoaffinity bead-based selection is also performed using anti-tumor or anti-epithelial cell antibodies attached to the beads. Although these procedures enrich HSCs and reduce tumor cell numbers, tumor cells can still be detected in the final product (Mapara, M. Y., Korner, I. J., Lentzsch, S., Krahl, D., Reichardt, P., and Dorken, B.: Combined positive/negative purging and transplantation of peripheral blood progenitor cell autografts in breast cancer patients: A pilot study. Exp.Hematol. 27: 169-175, 1999).
In another protocol, Clarke et al. disclosed the use of adenovirus-mediated transfer of suicide genes to selectively kill tumor cells (Clarke, M. F., Apel, I. J., Benedict, M. A., Eipers, P. G., Sumantran, V., Gonzalez-Garcia, M., Doedens, M., Fukunaga, N., Davidson, B., Dick, J. E., Minn, A. J., Boise, L. H., Thompson, C. B., Wicha, M., and Nunez, G.: A recombinant bcl-xS adenovirus selectively induces apoptosis in cancer cells but not in normal bone marrow cells. PNAS 92: 11024-11028, 1995). However, it is well known that virus-mediated gene transfer is far less than 100% efficient, which would result in a significant number of tumor cells being unaffected by the protocol.
Yet another method utilized fluorescence-activated cell sorting (FACS) to sort HSCs from tumor cells (Tricot, G., Gazitt, Y., Jagannath, S., Vesole, D., Reading, C. L., Juttner, C. A., Hoffman, R., and Barlogie, B.: CD34+Thy+linxe2x88x92 peripheral blood stem cells (PBSC) effect timely trilineage engraftment in multiple myeloma (MM). Blood 86: 293a-0, 1995). As is known, flow cytometry sorts cells one at a time and physically separates one population of cells from a mixture of cells based upon cell surface markers and physical characteristics. However, it has been shown that using FACS to separate large cell populations for clinical applications is not advantageous because the process is slow, the cell yields can be very low, and purity greater than xcx9c98% is rarely achieved.
Another method utilizing a flow cytometer is described in U.S. Pat. No. 4,395,397 to Shapiro. In the Shapiro method, labeled cells are placed in a flow cytometer, and a downstream laser beam is used to kill the labeled cells in the flowing stream after they pass by the detector and are recognized as being labeled by an electronic system. This method suffers from a number of disadvantages. Firstly, once an unwanted cell has passed through the detector/laser region there is no way to check that destruction has been completed successfully. If a tumor cell evades destruction it will inevitably be reintroduced into the patient. Secondly, the focal spot diameter of the laser beam is of necessity greater than the liquid stream cross section. Accordingly, many of the HSCs in the region of an unwanted cell will also be destroyed by the laser beam. Also, as described above, flow cytometric techniques do not provide pure samples of unlabeled cells due to the random and dynamic nature of the heterogeneous cell mixture that is flowing in a fast-moving (1-20 m/sec) stream of liquid.
Another method that utilizes laser technology is described in U.S. Pat. No. 4,629,687 to Schindler, et al. In this method, anchorage-dependent cells are grown on a movable surface. A small laser beam spot is scanned across the moving surface to illuminate cells one at a time and the information is then recorded. The same laser is then switched to a lethal power level, and the beam is swept over the surface in all areas except where a cell of interest was recorded during the illumination step. Unfortunately, this method is slow and only will work on cells that can adhere to a surface.
A still further method that utilizes laser technology is described in U.S. Pat. No. 5,035,693 to Kratzer. In this method, cells are placed on a moving belt and a small laser beam spot is scanned across the belt surface. When a particular cell radiates in response to the illuminating laser spot, the same laser is quickly switched to a high power setting in order to kill the cell in a near simultaneous manner before the laser has moved appreciably away from that cell. However, this system has many of the same disadvantages as the Shapiro method. For example, because the scanner is continuously moving during the imaging and killing process, the system is highly-dynamic, and therefore less stable and less accurate than a static system. Also, because the cells are moving on a belt past the detector in one direction, the method is not reversible. Thus, if a single tumor cell escapes detection, it will be reintroduced into the patient.
Others have used a small laser beam spot to dynamically scan over a surface to illuminate cells. For example, U.S. Pat. No. 4,284,897 to Sawamura et al. describes the use of galvanometric mirrors to scan a small laser beam spot in a standard microscope to illuminate fluorescent cells. U.S. Pat. No. 5,381,224 to Dixon et al. describes imaging of macroscopic specimens through the use of a laser beam spot that is raster-scanned with galvanometric mirrors through an F-theta scanning lens. In U.S. Pat. Nos. 5,646,411, 5,672,880, and 5,719,391 to Kain, scanning of a small laser spot with galvanometers through an F-theta lens is described. All of these imaging methods dynamically illuminate a small point that is moved over the surface to be imaged. In some cases, the surface being scanned also moves during imaging.
Similar methods of scanning a small laser spot have been described for purposes other than imaging of cells. For example, U.S. Pat. No. 4,532,402 to Overbeck describes the use of galvanometers to move a small laser beam spot over a semiconductor surface for repair of an integrated circuit. Similarly, U.S. Pat. No. 5,690,846 to Okada et al. describes laser processing by using mirrors to move a small laser spot through an F-theta scanning lens. U.S. Pat. No. 5,296,963 to Murakami et al. describes the use of galvanometric mirrors to scan a small laser beam spot in a standard inverted microscope to puncture cells for insertion of genetic matter.
Yet another method of scanning a biological specimen is described in U.S. Pat. No. 5,932,872 to Price. This method uses a plurality of detectors to simultaneously capture images at a plurality of focal planes from a constantly moving surface. The resultant images were used to choose the best-focus image in real-time in order to generate a three-dimensional volumetric image of a specimen.
The majority of methods described above are based on administering a tumor cell-removal or tumor cell-killing strategy to the entire harvested cell population as a whole. In flow cytometry, cells are sorted on a single cell basis to physically separate the unwanted tumor cells from HSCs. While each of these methods has been shown to reduce tumor cell numbers in HSC transplants, none has demonstrated the ability to remove or kill all detectable tumor cells. In fact, the majority of patient transplants still contain detectable tumor cells after these purging techniques are used. Approximately 30 to 30,000 tumor cells per transplant still remain, even after multiple-step purging procedures (Gazitt, Y., Reading, C. C., Hoffman, R., Wickrema, A., Vesole, D. H., Jagannath, S., Condino, J., Lee, B., Barlogie, B., and Tricot, G.: Purified CD34+linxe2x88x92Thy+ stem cells do not contain clonal myeloma cells. Blood 86: 381-389, 1995; Gribben et al. 1991; Mapara et al. 1997; Paulus et al. 1997).
Further, all of these methods result in some degree of HSC loss or damage, which can significantly impact the success of the HSC transplant by delaying patient engraftment. In summary, existing technologies for separating one cell population from another are inadequate, and there exists a great unmet clinical need for novel approaches that can effectively separate and treat one selected cell population within a second cell population. The method and apparatus described herein fulfills this need.
This invention provides a high-speed method and apparatus for selectively identifying, and individually targeting with an energy beam, specific cells within a mixed cell population of cells for the purpose of inducing a response in the targeted cells. Using the apparatus of the present invention, every detectable target cell in a mixed cell population can be specifically identified and targeted, without substantially affecting cells that are not being targeted.
Specific cells are identified with the disclosed invention using several approaches. One embodiment includes a non-destructive labeling method so that all of the target cells are substantially distinguishable from the non-target cells in the specimen. In this embodiment, a labeled antibody can be used to specifically mark each target cell, yet not mark non-target cells. The labeled cells are then identified within the cell mixture. A narrow energy beam is thereafter focused on the first of the targeted cells to achieve a desired response. The next of the targeted cells is then irradiated, and so on until every targeted cell has been irradiated.
In another embodiment, an antibody that selectively binds to non-target cells, but not to target cells, is used to identify the target cells by the absence of the label, and target cells are thereafter individually targeted with the energy beam.
To provide even greater flexibility in the ability to distinguish target cells from non-target cells, combinations of two or more labels, each with different fluorescent properties, can be used. For example, one antibody labeled with phycoerythrin (PE) and another antibody labeled with Texas Red(copyright) could be used to identify target cells that express one, both, or neither of the antigens recognized by the antibodies. One skilled in the art could propose the use of many multi-color labeling approaches to identify specific cell subpopulations within a complex mixture of cells.
The nature of the response that is induced by the energy beam is dependent upon the nature of the energy beam. Responses that can be induced with an energy beam include necrosis, apoptosis, optoporation (to allow entry of a substance that is present in the surrounding medium, including genetic material), cell lysis, cell motion (laser tweezers), cutting of cell components (laser scissors), activation of a photosensitive substance, excitation of a fluorescent reagent, etc.
One embodiment of the invention is an apparatus for selectively inducing a response in one or more targeted cells within a biological specimen that includes: an illumination source for illuminating a frame of cells in the biological specimen with a first wavelength of light; an image capture system that captures one or more images of the frame of cells; first commands for determining the locations of the one or more targeted cells within the frame of cells; an energy source that emits an energy beam sufficient to induce the response in at least one of the one or more targeted cells within the frame of cells; and second commands for steering the energy beam to the locations of the one or more targeted cells.
Still another embodiment of the invention is an apparatus for selectively inducing a response in one or more targeted cells within a biological specimen. This embodiment includes a first illumination source for illuminating a frame of cells in the biological specimen with a first wavelength of light; a second illumination source for illuminating a frame of cells in the biological specimen with a second wavelength of light; an image capture system that captures one or more images of the frame of cells; first commands for determining the locations of the one or more targeted cells within the frame of cells; an energy source that emits an energy beam sufficient to induce the response in at least one of the one or more targeted cells within the frame of cells; and second commands for steering the energy beam to the locations of the one or more targeted cells.
A still further embodiment is an apparatus for selectively inducing a first response in a first targeted cell population and a second response in a second targeted cell population within a biological specimen, providing: a means for illuminating a frame of cells in the biological specimen with one or more wavelengths of light; an image capture system that captures one or more images of the first targeted cell population and the second targeted cell population; first commands for determining the coordinates of one or more cells within the first targeted cell population and one or more cells within the second targeted cell population by reference to the captured one or more images; an energy source that emits an energy beam; and second commands for steering the energy beam to the coordinates.
One additional embodiment is an apparatus for monitoring the status of labeled cells within a biological sample. This embodiment includes: an illumination source for illuminating a population of cells in the biological sample so that the labeled cells are distinguishable from non-labeled cells; an image capture system that captures one or more images of the illuminated population of cells; first commands for determining the locations of labeled cells in the one or more images; a memory for storing the locations of the labeled cells in the biological sample; an energy source that emits a first energy beam to illuminate a first labeled cell; and a detector for measuring the fluorescence of the first labeled cell in response to illumination by the energy beam.
A further embodiment is a method for selectively inducing a response in one or more targeted cells within a biological specimen that includes: illuminating a frame of cells in the biological specimen with a first wavelength of light, wherein the biological specimen has been treated with one or more labels that, when excited by the first wavelength of light, emit energy at a one or more different wavelengths; capturing an image of the frame of cells; determining the locations of one or more targeted cells within the image by reference to cells that are activated by the first wavelength of light; and steering an energy beam to the locations of the one or more targeted cells, wherein the energy beam is sufficient to induce a response in at least one of the one or more targeted cells.
Yet a further embodiment is a method for inducing a first response in a first targeted population of cells and a second response in a second targeted population of cells, comprising: illuminating the first targeted population of cells and the second targeted population of cells with a first wavelength of light; capturing an image comprising the illuminated first targeted population of cells and the illuminated second targeted population of cells; determining the locations of the first targeted population of cells by reference to the image; determining the locations of the second targeted population of cells by reference to the image; and steering an energy beam to the locations of the first targeted population of cells and the second targeted population of cells in order to induce the first response in the first targeted population of cells and the second response in the second targeted population of cells.
A large number of commercially important research and clinical applications can be envisioned for such an apparatus, examples of which are presented below.